1. Field of the Invention
The present invention relates to a glucose sensor, more particularly a glucose sensor comprising a gel in which glucose oxidase is immobilized and a pH-sensing element.
2. Description of the Related Art
A variety of types of glucose sensors have been proposed to determine the concentration of glucose in a solution. The conventional techniques of immobilized enzyme electrode probes are shown in "Solid Phase Biochemistry" published by John Wiley & Sons, New York, 1983, page 479 to 505. Although many models of glucose sensors have been proposed, their common principle for measuring the concentration of glucose is based on the oxidation reaction of glucose which is catalyzed by glucose oxidase (abbreviated as GOD hereinafter).
In the glucose sensor, oxygen (O.sub.2) consumed by the oxidation reaction of glucose, and hydrogen peroxide (H.sub.2 O.sub.2) and gluconic acid which are produced by the oxidation reaction of glucose are sensed or measured by means of electrochemical elements to determine the concentration of glucose. The electrochemical elements may consist of a pH glass electrode or a hydrogen-ion-sensitive field effect transistor (abbreviated as pH-ISFET, hereinafter) which detects pH-change or pH-variation caused by the production of gluconic acid.
An example of the pH glass electrode is disclosed in "Biochimica et Biophysica Acta," Nilsson et al., 1973, vol 320, page 529, while a variety of models of the glucose sensors using the pH-ISFET have been proposed recently, for example "IEEE Transactions on Electron Devices," 1986, vol. ED-33, No. 1, page 47. In these conventional glucose sensors, a gel containing a GOD, which is available on the market, is held or deposited on a pH-sensing region of the pH-ISFET.
In the pH-change sensing type of enzymatic glucose probes above-mentioned, protons dissociated from gluconic acid produced by hydrolysis of D-glucono-.delta.-lactone, which is a direct product of the oxidation reaction of glucose are detected.
Gluconic acid possesses a dissociation constant (pKa) lower than 4 and hence is dissociated almost completely into a proton and a conjugate base at a pH range higher than 6. On the other hand, the D-glucono-.delta.-lactone which is produced by the oxidation reaction of glucose is almost completely hydrolyzed at a pH range higher than 5, since equilibrium in the hydrolysis reaction of the D-glucono-.delta.-lactone at the pH range higher than 5 is shifted excessively to the side of gluconic acid formation.
Therefore, if the pH is higher than 6, the D-glucono-.delta.-lactone which is a direct product of the oxidation reaction of glucose is hydrolyzed almost completely into gluconic acid which in turn is dissociated almost completely into a proton and a conjugate base, so that it can be considered that there is a relationship of 1:1 between the amount of glucose which is consumed by the oxidation reaction of glucose and the amount of protons which are detected. In the pH-change sensing type of enzymatic glucose probes, since the pH-change created on the pH sensing region is detected through the above-mentioned reaction scheme it is preferable that the hydrolysis reaction of D-glucono-.delta.-lactone proceed as fast as possible and also that the consumption rate of glucose and the formation rate of gluconic acid are balanced.
However, the rate constant of reaction in the hydrolysis of D-glucono-.delta.-lactone is in the order of 10.sup.-3 sec.sup.-1 (Y. Pocker et al., "Journal of American Chemical Society," 1973, vol 95, page 113) in the case of spontaneous hydrolysis reaction. This means that it takes more than 10 minutes to hydrolyze half of the amount of D-glucono-.delta.-lactone at ambient temperature and at neutral pH.
Therefore, in the pH-change sensing type of enzymatic glucose probes in which the oxidation reaction of glucose proceeds in the gel containing the reference substance GOD, if the velocity of the hydrolysis of D-glucono-.delta.-lactone is slow, the greater portion of the D-glucono-.delta.-lactone which is produced by the oxidation reaction of glucose disappears or is lost out of the gel before it is converted to gluconic acid, so that the portion which disappears or is lost does not contribute to pH-change on the pH-sensing region of the glass pH-electrode or of the pH-ISFET.
In fact, it was confirmed by the present inventor that no response to glucose was observed in a case where GOD of high purity (GI obtained from ORIENTAL KOBO Co., Ltd. and Buhlinger Mannheim) was used in an pH-ISFET glucose sensor which was constructed according to a method disclosed in Japanese Patent Application No. 59-209165 (Laid-Open No. 61-88135, published May 6, 1986), even if the buffer capacity of a solution to be measured was lowered to the order of 0.002. This means that it is necessary to accelerate hydrolysis of D-glucono-.delta.-lactone in the pH-change sensing type of enzymatic glucose probes.
In the course of a study which was conducted to solve such problems, it was found that some of the GOD available on the market exhibited gluconolactonase (EC 3.1.1.17) activity which accelerates the hydrolysis of D-glucono-.delta.-lactone and that the out-put of the pH-ISFET glucose sensors containing such gluconolactonase were as high as the same level of those that had been reported.
Although it was already reported in "Biotechnology and Bioengineering," 1977, Vol. 19, page 185, that gluconolactonase is contained in commercially available GOD, this paper relates to reactions for preparing gluconic acid from maltose but mentions nothing about utilization of gluconolactonase in the pH-change sensing type of enzymatic glucose sensors.
The present inventor found that the gluconolactonase can be used as an accelerator of the hydrolysis of D-glucono-.delta.-lactone and completed present invention.
Therefore, an object of the present invention is to overcome the problem of the prior arts above, mentioned by accelerating the hydrolysis of D-glucono-.delta.-lactone and to provide an improved glucose sensor whose out-put signals are sufficiently high and hence is applicable for practical uses even at low concentrations of glucose.